BK virus (BKV)-specific immunity is critical for polyomavirus-associated nephropathy, but antibody responses are incompletely defined. We compared the hemagglutination inhibition assay (HIA) with immunoglobulin G enzyme immunoassays (EIA) to BKV proteins expressed in baculovirus-infected insect cells. N-terminal, internal, and C-terminal domains of the BKV large T antigen (BKLT) were fused to glutathione S-transferase (GST), yielding GST-BKLTD1, GST-BKLTD2, and GST-BKLTD3, respectively. The BKV capsid VP1 was expressed as a GST fusion (BKVP1) or as a native VP1 assembled into viruslike particles (BKVLP). We tested 422 sera from 28 healthy donors (HD), 99 dialysis patients (DP; median age, 15 years; range, 3 to 32 years), and 46 age-matched kidney transplant patients (KTP; median age, 15 years; range, 2 to 33 years). In HD, HIA and BKVLP EIA both yielded a 91.7% seroreactivity, whereas all other EIA responses were lower (BKVP1, 83.3%; BKLTD1, 25%; BKLTD2, 29%; BKLTD3, 40%). HIA titers significantly correlated with EIA levels for BKVLP, BKVP1, and BKLTD1 but not for BKLTD2 or BKLTD3, which were barely above the cutoff. In DP, the seroreactivities of HIA, BKVLP, and BKLTD1 were lower than that in HD (63.6%, 86.9%, and 10.1%, respectively) and they had lower titers (P < 0.001). In KTP, seropositivities for BKVLP, BKVP1, and BKLTD1 were 78%, 50%, and 17%, respectively, but anti-BKVLP levels increased significantly in KTP with viruria and viremia, whereas anti-BKLTD1 levels increased after clearing sustained BKV viremia. In conclusion, anti-BKVLP is equivalent to HIA in HD but is more sensitive to determine the BKV serostatus in DP and KTP. In KTP, anti-BKVLP responds to recent BKV viruria and viremia, whereas anti-BKLTD1 may indicate emerging BKV-specific immune control.Polyomaviruses (PyV) have been identified in many vertebrates; the main species found in humans are BK virus (BKV) and JC virus (C. Büchen-Osmond, Polyomaviridae, International Committee on Taxonomy of Viruses Database [www .ncbi.nlm.nih.gov/ICTVdb/]). PyV are small nonenveloped double-stranded DNA viruses with icosahedral particles of ϳ42 nm in diameter and are resistant to environmental inactivation (8, 31, 32; www.ncbi.nlm.nih.gov/ICTVdb/). The genome organization of the 5.1-kb circular genome is largely conserved and encodes six major proteins (20, 31, 32). The nuclear large tumor (LT) antigen and cytoplasmic small T antigen are expressed early in the viral life cycle, followed by the late cytoplasmic agnoprotein and the capsid proteins VP1, VP2, and VP3, which are transported into the nucleus for virion assembly (31,32,44). LT is a multifunctional regulatory protein with distinct domains (45,49). PyV capsids are formed by the assembly of 72 VP1 pentamers into a Tϭ7d icosahedral lattice associating with VP2 and VP3 and the circular viral genome (11,35,39).Seroprevalence data indicate that 50 to 90% of the general population has been exposed to BKV and JC virus (25,33,47).Both viruses persist in the renourinary tract as the principal site of latenc...

Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66 were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function.

Objective The current Ebola epidemic massively affected the Macenta district in Forest Guinea. We aimed at investigating its impact on general and HIV care at the only HIV care facility in the district. Design Prospective observational single-facility study Methods Routinely collected data on use of general hospital services and HIV care were linked to Ebola surveillance data published by the Guinea Ministry of Health. In addition we compared retention among HIV-infected patients enrolled into care in the first semesters of 2013 and 2014. Results Throughout 2014, service offer was continuous and unaltered at the facility. During the main epidemic period (August – December 2014), compared to the same period of 2013, there were important reductions in attendance at the primary care outpatient clinic (−40%), in HIV tests done (−46%), in new diagnoses of tuberculosis (−53%), in patients enrolled into HIV care (−47%). There was a smaller reduction in attendance at the HIV follow-up clinic (−11%). Kaplan-Meier estimates of retention were similar among the patients enrolled into care in 2014 and 2013. In a multivariable Cox regression analysis, the year of enrolment was not associated with attrition (hazard ratio 1.02; 95% confidence interval: 0.72–1.43). Conclusions The Ebola epidemic resulted in an important decrease of utilization of the facility despite unaltered service offer. Effects on care of HIV-positive patients enrolled prior to the epidemic were limited. HIV care in such circumstances is challenging, but not impossible.

Virus replication and progression to disease in transplant patients is determined by patient-, graftand virus-specific factors. This complex interaction is modulated by the net state of immunosuppression and its impact on virus-specific cellular immunity. Due to the increasing potency of immunosuppressive regimens, graft rejections have decreased, but susceptibility to infections has increased. Therefore, cytomegalovirus (CMV) remains the most important viral pathogen posttransplant despite availability of effective antiviral drugs and validated strategies for prophylactic, preemptive and therapeutic intervention. CMV replication can affect almost every organ system, with frequent recurrences and increasing rates of antiviral resistance. Together with indirect long-term effects, CMV significantly reduces graft and patient survival after solid organ and hematopoietic stem cell transplantation. The human polyomavirus called BK virus (BKV), on the other hand, only recently surfaced as pathogen with organ tropism largely limited to the reno-urinary tract, manifesting as polyomavirus-associated nephropathy in kidney transplant and hemorrhagic cystitis in hematopoetic stem cell transplant patients. No licensed anti-polyoma viral drugs are available, and treatment relies mainly on improving immune functions to regain control over BKV replication. In this review, we discuss diagnostic and therapeutic aspects of CMV and BKV replication and disease posttransplantation.

Impaired BK virus (BKV)-specific immunity is a key risk factor of polyomavirus-associated nephropathy. We hypothesized that BKV agnoprotein might constitute an important immune target, as it is highly expressed after infection in vitro. We demonstrate abundant expression of BKV agnoprotein in vivo by immunostaining of kidney transplant (KT) biopsy specimens. Antibody responses to the recombinant affinity-purified BKV agnoprotein, large tumor (LT), and VP1 antigens in 146 sera from 38 KT patients and in 19 sera from 16 healthy donors (HD) were compared by enzyme immunoassay. In HD, low titers of anti-agnoprotein immunoglobulin G (IgG) were found in 15% of sera, compared to 41% for anti-LT antigen and 63% for anti-VP1. No anti-BKV IgM was detectable. In KT patients, anti-agnoprotein IgG and IgM were found in 8% and 3.6% of sera, compared to 63% and 18% for anti-LT IgG and IgM and 80% and 41% for anti-VP1 IgG and IgM, respectively. Anti-LT antigen and anti-VP1, but not anti-agnoprotein, activities increased during and after BKV viremia in KT patients. To investigate specific cellular immune responses, we compared levels of gamma interferon production in peripheral blood mononuclear cells (PBMC) of 10 HD and 30 KT patients by enzyme-linked immunospot assay. In HD, the median numbers of gamma interferon spot-forming units per million PBMC for the agnoprotein, LT antigen, and VP1 peptides were 1, 23, and 25, respectively, whereas the responses in KT patients were 2, 24, and 99, respectively. We conclude that BKV agnoprotein, though abundantly expressed in vivo, is poorly recognized immunologically.The human polyomavirus BK virus (BKV) is the primary etiological agent of polyomavirus-associated nephropathy (PVAN), which causes irreversible graft loss in 1 to 10% of kidney transplant (KT) patients (15, 31). BKV was first discovered in 1970 in the urine of a KT patient with the initials B.K. who had ureteric stenosis and abundant decoy cell shedding (9). However, BKV asymptomatically infects 60 to 90% of the human population (23) and establishes a state of nonreplicative infection in the renourinary tract (13, 15). Intermittent low-level urinary replication with BKV loads of Յ10e6 per ml is detected in 5% of immunocompetent individuals, whereas high-level replication with BKV loads of Ն10e7 per ml is found in 20 to 60% of immunosuppressed patients (15). In KT patients, high-level urine BKV replication is found in 30%, which may be followed by BKV viremia in 13% and by histologically confirmed PVAN in 8% of patients (18). The risk factors for PVAN are not conclusively defined and likely involve complementing determinants of the triad of recipient, graft, and virus (17). Disruption of the balance between the BKV replication and host immune control is generally viewed as a key element of PVAN pathogenesis (5). In the absence of validated antivirals, reducing maintenance immunosuppression represents the primary treatment option for presumptive or definitive PVAN (3,39).BKV belongs to the genus Polyomavirus of the Polyomavir...